Regulated intracellular proteolysis controls virtually every aspect of cell physiology. The process is highly selective, with individual protein half-live ranging from minutes to weeks. This selectivity is ensured by specific interactions between substrates and E3 ubiquitin ligase complexes, which mark proteins for degradation with a chain of ubiquitin moieties. Many E3 ligases are modular, based on a core scaffold, with interchangeable substrate-targeting subunits, which enables one piece of core machinery to ubiquitylate many different substrates. The cullin-RING ligase (CRL) family of complexes are the archetypes for these modular ubiquitin ligases, and CRL1 ligases, better known as the SKP1-CUL1-F-box protein (SCF) complexes, are the best characterized. SCFs use a family of F-box proteins (69 in humans) as substrate adaptors to mediate the ubiquitylation and consequent degradation of a large number of regulatory proteins involved in diverse processes. During the initial years of GM57587, my laboratory investigated how two SCF complexes (SCF- SKP2 and SCF-TrCP) and APC/C (a CRL-like ubiquitin ligase) target various regulators of cyclin dependent kinases (CDKs) for degradation. Subsequently, to broaden our knowledge of these remarkable enzymes, we asked whether any other members of the F-box protein family play important roles in controlling the cell division cycle. For example, during the last funding cycle, we found that cyclin F (FBXO1) controls genome integrity by coordinating production of deoxyribonucleotides with DNA replication; FBH1 (FBXO18) promotes DNA double strand breakage and apoptosis in response to DNA replication stress; and FBXO11 regulates cell quiescence and differentiation. We now propose a project exploring the integration of CRL- controlled cell cycle networks with DNA replication and the DNA damage response. Via proteomic techniques, we have identified putative CRL substrates involved in these processes, and using an interdisciplinary approach that incorporates biochemistry, molecular cell biology, and somatic cell genetics, we will characterize the molecular mechanisms and biological significance of the degradation of these novel substrates.